Heparin is an acid mucopolysaccharide found in the liver, intestines, lungs, skin, and the like, and contains sulfated D-glucosamine, D-glucuronic acid, L-iduronic acid, and the like.
Heparin has a strong anticoagulant activity, and is hence used to treat disseminated intravascular coagulation syndrome (DIC), treat and prevent various thromboembolisms (venous thrombosis, myocardial infarction, pulmonary embolism, cerebral embolism, thromboembolism in upper and lower extremities, pre/post operation thromboembolism, or the like), prevent the blood coagulation at the occasions of extracorporeal circulation apparatus use for haemodialysis, artificial heart and lung, or the like, vessel catheter insertion, blood transfusion, blood tests, or the like.
Further, heparin is known to have many physiological activities, in addition to the anticoagulant activity, such as lipoprotein lipase activation action, antiplatelet aggregation action, hypotensive action, anticomplementary action, cancer metastasis inhibitory action, inhibitory effect on degranulation from mast cells, local inflammation suppression, analgesic action and blood circulation improving action on muscle tissues, and the like.
Heparin is produced by the extraction/fractionation from tissues of principally healthy edible animals, but, ever since the BSE (bovine spongiform encephalopathy) incidence, heparin used as pharmaceutical products is mostly originated from porcine intestinal mucosa. Typically, the porcine intestinal mucosa is suspended in an aqueous solvent for proteolytic digestion, and subsequently an adsorbent, or the like, (Non Patent Literature 1), is added thereto to extract heparin and other mucopolysaccharides (principally chondroitin sulfate family, heparan sulfate, and the like) as a complex to use as a crude material. Then, the crude material is batch mixed/fractionated to obtain heparin (so-called “unfractionated heparin”).
The heparin (unfractionated heparin) obtained by the above method contains mucopolysaccharides (mainly heparan sulfate, chondroitin sulfates B and C) other than the heparin, and it is known that the content thereof varies depending on the crude material and production method. However, the side effects caused by those impurities are roughly confirmed and accepted, and as a result, the unfractionated heparin has been used as a pharmaceutical product for many years.
However, in the early 2008, there were many cases reported in the U.S. and Germany that patients administered with an unfractionated heparin preparation by bolus injection (rapid intravenous injection) experienced unusual side effects, and the incidence eventually caused more than 80 deaths. The US FDA analyzed the unfractionated heparin preparation and the bulk drug thereof and confirmed that they were evidently different from the conventional products, and further revealed that oversulfated chondroitin sulfate (OSCS) was identified to have been present in them (Non Patent Literatures 2 to 4). This does not exist as such in nature, and is believed to have been most likely mixed in at the time of bulk drug production.
Serious side effects were not reported in Japan, but a part of the unfractionated heparin preparations and low molecular weight heparin (LMWH) preparations were recalled which caused serious problems in the stable supply to the market.
Many researchers have been studying on OSCS for scientific synthesis methods, the structure analysis, investigation to determine the causes of adverse events by animal tests, or the like, particularly studying on the purity and safety assessment by 1H-NMR, other test methods, or the like, (Non Patent Literatures 5 and 6), and OSCS has been specified as the causative substance of adverse events in Japan and thus safety/purity tests have been reviewed by administration agencies and preparation manufacturers (Non Patent Literatures 7 and 8).
Meanwhile, the methods described in 1) Non Patent Literature 1, 2) Non Patent Literature 9, 3) Non Patent Literature 10, 4) Patent Literature 1, and the like, are known for producing or purifying heparin.
However, there has been no method known for easily and effectively removing impurities such as OSCS, chondroitin sulfate, and the like, from heparin. Further, there has been no method known for easily detecting or measuring such impurities in heparin.